Ben-David, Ateret2023-02-202023-02-202022https://hdl.handle.net/10539/34594A dissertation submitted in fulfilment of the requirements for the degree of Master of Science to the Faculty of Science, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, 2022Nitrile hydratases (NHase) are important enzymes that can transform nitriles, which are low cost intermediates, into high-value industrial chemicals like acrylamide. To increase the use of NHase as a biocatalyst in industry, it needs to be engineered to work in harsher conditions. A high-throughput screening assay must be developed before indirect evolution can be conducted so that enzyme activity of the mutants can be determined. In this study, a coupled-enzyme assay was employed to convert the amide generated by the NHase to hydroxamic acid, which could then be colorimetrically identified. The NHase was successfully transformed and expressed prior to assay optimisation. Multiple steps were engaged in the optimisation process, including examining the influence of substrate and product inhibition on the NHase and amidase, as well as the effect of temperature, pH, and organic solvent on the assay. The optimal conditions were determined to be 100 mM acetonitrile and 1.0 µg/µl NHase which were incubated for 10 minutes. After which 0.4 units of amidase and 20 mM hydroxylamine were added to the well and incubated for a further 30 minutes. The optimal incubation temperature and pH were determined to be 35˚C and pH 8. The study resulted in a sensitive, high-throughput screening assay for NHase enzymes.enDissertation